Lyme disease, the most prevalent arthropod-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. Despite intensive study in recent years little is known regarding the pathogenesis of infection at the molecular level. We have determined the genetic diversity among clinical isolates of B. burgdorferi and shown that spirochete dissemination varies significantly in patients and mice infected with distinct genotypes. These results demonstrate that different genotypes of B. burgdorferi possess varying potential for dissemination in an infected host. We hypothesize that these differences are the result of variations in gene content and/or expression among different genotypes. We propose to employ functional genomics and proteomics to gain insight into possible differences in gene/protein expression between B. burgdorferi strains with differing capacities for hematogenous dissemination. The long-term objective of this project is elucidation of genes and/or proteins that mediate B. burgdorferi virulence. The following specific aims are proposed: 1) global gene expression of B. burgdorferi clinical isolates of differing genotype in varying environments will be monitored by gene array; 2) differences in protein expression among the various genotypes will be assessed by proteomics; 3) the roles of candidate virulence genes in pathogenesis of Lyme disease will be evaluated by monitoring expression for a number of the most promising candidate genes will be monitored in early Lyme disease patients and infected mice by real-time RT-PCR; 4) selected candidate virulence genes will then be targeted for insertional inactivation and complementation. The effects of such genetic manipulation should provide direct demonstration of the critical role such genes may play in spirochete pathogenesis. \The combination of functional genomics, in vivo and genetic approaches proposed here should provide for the comprehensive assessment of the molecular basis of pathogenesis for B. burgdorferi isolates with varying potential for bloodstream dissemination and result in identification of determinants required for spirochete dissemination. This will provide new insights into the natural history of B. burgdorferi infection in humans, which, in turn, may have implications for prevention, treatment, and diagnosis of Lyme disease.